Fig. 6. Effect of cytoskeleton-perturbing agents on secretion (A) and Munc18-2 relocation (B). (A) RBL cells were seeded in 96-well plates and sensitised overnight with IgE. Cells were treated with the indicated concentrations of either cytochalasin D for 30 minutes or nocodazole for 1 hour, or corresponding vehicles prior to stimulation with DNP-HSA. Released SG content was determined by ß-hexosminidase release. (B) RBL cells seeded on coverslips were incubated with either cytochalasin D (10 µM) for 1 hour, nocodazole (33 µM) for 1 hour or EGTA (5 mM) for 15 minutes before stimulation with DNP-HSA for 10 minutes. Cells were processed for confocal analysis and probed with antibodies to Munc18-2 (red) and either phalloidin-FITC (green) or antibody to -tubulin (green) as before. Cells were visualised by confocal microscopy as described in Materials and Methods. The merged images (green and red fluorescence) of several, as well as single cells are shown. As after treatment with cytochalasin D phalloidin staining is decreased, presentation of an overlay of the DIC image with Munc18-2 staining is preferred. Note also that after nocodazole-treatment MT staining either disappears (single cell panel) or is severely restricted to the MTOC. Bars, 5 µm. Images are representative of at least three experiments.

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